Nerium oleander is an ornamental plant widely distributed in subtropical Asia, the southwestern United States, and the Mediterranean. Its medical and toxicological properties have long been recognized. It has been used, for example, in the treatment of hemorrhoids, ulcers, leprosy, snake bites, and even in the induction of abortion. Oleandrin, an important component of oleander extract, is a potent inhibitor of human tumor cell growth (Afaq F et al. Toxicol. Appl. Pharmacol. 195:361-369, 2004). Oleandrin-mediated cell death is associated with calcium influx, release of cytochrome C from mitochondria, proteolytic processes of caspases 8 and 3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation.
It has been demonstrated that oleandrin is the principal cytotoxic component of Nerium oleander (Newman, et al., J. Herbal Pharmacotherapy, vol. 13, pp. 1-15, 2001). Oleandrin is a cardiac glycoside that is exogeneous and not normally present in the body. Oleandrin induces apoptosis in human but not in murine tumor cell lines (Pathak et al., Anti-Cancer Drugs, vol. 11, pp. 455-463, 2000), inhibits activation of NF-kB (Manna et al., Cancer Res., vol. 60, pp. 3838-3847, 2000), and mediates cell death in part through a calcium-mediated release of cytochrome C (McConkey et al., Cancer Res., vol. 60, pp. 3807-3812, 2000). A Phase I trial of a hot water oleander extract has been completed recently (Mekhail et al., Am. Soc. Clin. Oncol., vol. 20, p. 82b, 2001). It was concluded that oleander extracts can be safely administered at doses up to 1.2 ml/m2/d. No dose limiting toxicities were found.
In addition to being selectively cytotoxic for tumor cells, cardiac glycosides may also enhance cell response to cytotoxic actions of ionizing radiation. Ouabain, a cardiac glycoside endogeneous to the body, was reported to enhance in vitro radiosensitivity of A549 human lung adenocarcinoma cells but was ineffective in modifying the radioresponse of normal human lung fibroblasts (Lawrence, Int. J. Radiat. Oncol. Biol. Phys., vol. 15, pp. 953-958, 1988). Ouabain was subsequently shown to radiosensitize human tumor cells of different histology types including squamous cell carcinoma and melanoma (Verheye-Dua et al., Strahlenther. Onkol., vol. 176, pp. 186-191, 2000). Although the mechanisms of ouabain-induced radiosensitization are still not fully explained, inhibition of repair from sublethal radiation damage and an increase in radiation-induced apoptosis have been advanced as possibilities (Lawrence, 2000; Verheye Dua et al., 2000; Verheye-Dua et al., Strahlenther. Onkol., vol. 172, pp. 156-161, 1996). The cardiac glycoside oleandrin also has the ability to enhance the sensitivity of cells to the cytotoxic action of ionizing radiation. See U.S. patent application Ser. No. 10/957,875 to Newman, et al. and Nasu et al., Cancer Lett. Vol 185, pp. 145-151, 2002).
Chen et al. (Breast Cancer Research and Treatment (2006), 96, 1-15) suggest that cardiac glycosides, such as ouabain and digitalis, might be useful toward developing anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.
Smith et al. (Biochemical Pharmacology (2000), 62, 1-4) report that ANVIRZEL, and its key cardiac glycoside component oleandrin, inhibits the exportation of fibroblast growth factor-2 (FGF-2) from the prostate cancer cell lines PC3 and DU145.
Newman et al. (J. Experimental Therapeutics and Oncology (2006), 5, 167-181) report that incubation of human malignant melanoma BRO cells with oleandrin results in a time-dependent formation of reactive oxygen species, superoxide anion radicals, that mediate mitochondrial injury and loss of cellular GSH pools.
U.S. Pregrant Patent Application Publication No. 20050112059 to Newman et al. discloses the enhancement of radiotherapy in the treatment of cancer by administration of oleandrin.
Extraction of glycosides from plants of Nerium species has traditionally been carried out using boiling water. The process of using boiling water as an extraction method to obtain active ingredients from Nerium oleander yields many products. Among these are oleandrin, nerine, and other cardiac glycoside compounds. The plant extracts are useful in the treatment of cell-proliferative diseases in animals.
Oleandrin extracts obtained by hot-water extraction of Nerium oleander, sold under the trademark ANVIRZEL™, are commercially available and contain the concentrated form or powdered form of a hot-water extract of Nerium oleander. The extract is prepared according to the process developed by Dr. Huseyin Ziya Ozel. U.S. Pat. No. 5,135,745 describes a procedure for the preparation of the extract of the plant in water. The extraction of the plant Nerium oleander involves slicing the leaves, cooking the sliced leaves and stems of the plant in water for 2-3 hours and filtering off the residues. The mixture is heated again. The aqueous extract reportedly contains several polysaccharides with molecular weights varying from 2 KD to 30 KD, oleandrin and oleandrigenin, odoroside and neritaloside. The polysaccharides reportedly include acidic homopolygalacturonans or arabinogalaturonans. Ozel (ibid, and Planta Med. 56 (1990), 668) incorrectly reports that the cardiac glycosides are not responsible for the antitumor activity of the aqueous extract.
Muller et al. (Pharmazie. (1991) September 46(9), 657-663) disclose the results regarding the analysis of a water extract of Nerium oleander. They report that the polysaccharide present is primarily galacturonic acid. Other saccharides include rhamnose, arabinose and galactose. Polysaccharide content and individual sugar composition of polysaccharides within the hot water extract of Nerium oleander have also been reported by Newman et al. (J. Herbal Pharmacotherapy, (2001) vol 1, pp. 1-16).
U.S. Pat. No. 5,869,060 to Selvaraj et al. pertains to extracts of Nerium species and methods of production. To prepare the extract, plant material is placed in water and boiled. The crude extract is then separated from the plant matter and sterilized by filtration. The resultant extract can then be lyophilized to produce a powder.
U.S. Pat. No. 6,565,897 (U.S. Pregrant Publication No. 20020114852 and PCT International Publication No. WO 2000/016793 to Selvaraj et al.) discloses a hot-water extraction process for the preparation of a substantially sterile extract.
Erdemoglu et al. (J. Ethnopharmacol. (2003) November 89(1), 123-129) discloses results for the comparison of aqueous and ethanolic extracts of plants, including Nerium oleander, based upon their anti-nociceptive and anti-inflammatory activities.
Organic solvent extracts of Nerium oleander are also disclosed by Adome et al. (Afr. Health Sci. (2003) August 3(2), 77-86; ethanolic extract), el-Shazly et al. (J. Egypt Soc. Parasitol. (1996), August 26(2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999) February 50(3), 435-438; methanolic extract), Zia et al. (J. Ethnolpharmacol. (1995) November 49(1), 33-39; methanolic extract), and Vlasenko et al. (Farmatsiia. (1972) September-October 21(5), 46-47; alcoholic extract).
Supercritical fluid extraction involves the use of a supercritical fluid to selectively extract a particular compound. A supercritical fluid is a liquid or a gas at atmospheric conditions, but becomes supercritical when it is compressed above its critical pressure and heated above its critical temperature. Supercritical fluids have increased dissolving power in their supercritical regions. A supercritical fluid exhibits properties between those of a gas and a liquid, and has the capacity to dissolve compounds that may only dissolve poorly or not at all in the gas or liquid state. Supercritical fluids are ideal for extraction of these compounds because they have high dissolving power at high densities and demonstrate good fractionation and separation of the compound from the fluid at lower densities when the pressure or temperature is changed. The general procedure of using supercritical carbon dioxide extraction in food processing industry has been described by Raventos, et al., in 2002 (M. Raventos, et al., Application and Possibilities of Supercritical CO2 Extraction in Food Processing Industry: An Overview, Food Sci. Tech. Int. Vol. 8 (5) (2002) 269-284), the entire content of which is hereby incorporated by reference. Raventos et al. report that SCF extraction is not suitable for extraction of polysaccharides from plant material even though he suggests that a modifier such as ethanol may be used to modify the performance of an SCF extraction.
U.S. Pregrant Patent Application Publication No. 20040247660 to Singh et al. discloses the preparation of a protein stabilized liposomal formulation of oleandrin for use in the treatment of cancer.
U.S. Pregrant Patent Application Publication No. 20050026849 to Singh et al. discloses a water soluble formulation of oleandrin containing a cyclodextrin. The '849 Publication suggests the preparation of solid-filled capsules containing the cyclodextrin complex of oleandrin. The oleandrin has been provided as the hot-water extract or the chemical entity and then treated with the cyclodextrin to form the complex.
U.S. Pregrant Patent Application Publication No. 20040082521 to Singh et al. discloses the preparation of protein stabilized nanoparticle formulations of oleandrin from the hot-water extract. The nanoparticles are prepared via formation of a liposomal mixture and subsequent evaporation of the organic solvent therein.
Methods to enhance the relative content of oleandrin from plant material are therefore warranted. While hot water extracts of Nerium oleander may provide oleandrin and related cardiac glycosides in relatively low yield, an improved method for obtaining a concentrated form of cardiac glycosides including oleandrin is needed.
Oleandrin contains a lactone ring that is acid labile and predisposes the material to acid degradation when orally dosed, so care must be taken in the preparation of liquid formulations to ensure minimization of acidic species in solution.
None of the known art discloses a pharmaceutical formulation comprising an extract of Nerium species, in particular, Nerium oleander. None of the art discloses or suggests a supercritical fluid extract comprising a cardiac glycoside, such as oleandrin. A need remains for more dosage forms that provide suitable delivery of the components of an extract of Nerium species for the treatment of various diseases and disorders. A need also remains for improved processes for obtaining cardiac glycosides by extraction from plant material.